CN114829395A

CN114829395A - anti-ANGPTL 3 antibodies and uses thereof

Info

Publication number: CN114829395A
Application number: CN202180007277.5A
Authority: CN
Inventors: 付雅媛; 许迎霞; 林冰; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2020-01-22
Filing date: 2021-01-22
Publication date: 2022-07-29
Anticipated expiration: 2041-01-22
Also published as: EP4095157A4; BR112022014306A2; US20230138315A1; EP4095157A1; WO2021147984A1; CA3166346A1; MX2022008471A; KR20220131935A; TW202140547A; AU2021209746A1; JP2023510787A; CN114829395B

Abstract

Relates to anti-ANGPTL 3 antibodies and uses thereof. In particular to an anti-ANGPTL 3 antibody and an antigen binding fragment thereof, or a pharmaceutically acceptable salt or a solvent compound thereof, and application thereof as a medicament, especially application thereof in preparing a medicament for treating hyperlipidaemia.

Description

anti-ANGPTL 3 antibodies and uses thereof

Technical Field

The present disclosure relates to the field of antibody pharmaceuticals. Specifically, the anti-ANGPTL 3 antibody drug and the application thereof are included.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

The angiopoietin-like protein 3(ANGPTL3, ANGPTL-3) gene is a novel gene identified by Conklin et al from EST databases based on signal sequence and amphipathic helical structure, which isolated the full-length cDNA of human ANGPTL3 from a human embryonic liver/spleen cDNA library (Conklin et al, 1999, Genomics 62: 477-482). The human ANGPTL3 protein (hANGPTL3) consisting of 460 amino acids shares 76% amino acid sequence identity with the mouse ANGPTL3 protein and has the characteristic structure of angiogenin, including a signal peptide, an extended helical domain expected to form a dimeric or trimeric coiled-coil, a short linker peptide, and a globular fibrinogen homologous domain (FD) (Conklin et al, 1999, Genomics 62: 477-482). ANGPTL3 differs from Angiopoietins (ANGS), ANGPTL3 does not bind to Tie2, but it also induces angiogenesis by binding to integrin α v β 3 via its C-terminal FD (Camenisch et al, 2002, J Biol Chem277: 17281-17290).

In addition, ANGPTL3 is an important factor affecting fat metabolism, ANGPTL3 inhibits LPL activity, thereby reducing clearance of triglycerides and low density lipoproteins, and hypertriglyceridemia is involved in the pathogenesis of a range of metabolic diseases. Elevated plasma triglyceride levels are implicated in the pathological process of atherosclerosis by inducing platelet aggregation and the production of plasminogen activator inhibitor-1 (PAI-1), as well as increasing the number of foam cells and promoting the proliferation and migration of vascular smooth muscle cells. The anti-ANGPTL 3 antibody can be specifically combined with ANGPTL3, so that the activity of ANGPTL3 is inhibited or interfered, and diseases such as atherosclerosis and hyperlipidemia are further treated. Currently, patent documents such as WO2012174178a1, WO2008073300a2, and CN107085112A disclose various anti-ANGPTL 3 antibodies.

Disclosure of Invention

The present disclosure provides an anti-ANGPTL 3 antibody or antigen binding fragment thereof. In some embodiments, the present disclosure provides an anti-ANGPTL 3 antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region comprises a heavy chain variable region and a heavy chain variable region as set forth in SEQ ID NO:9 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 10 having the same sequence as LCDR1, LCDR2 and LCDR 3;

ii) the heavy chain variable region comprises a sequence identical to the sequence set forth in SEQ ID NO:11 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising an amino acid sequence identical to that shown in SEQ ID NO: 12, the light chain variable region shown in the sequence has LCDR1, LCDR2 and LCDR3 with the same sequence; or

iii) the heavy chain variable region comprises a heavy chain variable region substantially identical to the heavy chain variable region as set forth in SEQ ID NO:13 having the same sequence as HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: the light chain variable region shown in the 14 sequence has LCDR1, LCDR2 and LCDR3 with the same sequence.

In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, wherein:

iv) the heavy chain variable region comprises the sequences shown in SEQ ID NO: 18. SEQ ID NO: 19 and SEQ ID NO: 20 and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 15. the amino acid sequence of SEQ ID NO: 16 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 17;

v) the heavy chain variable region comprises the sequences shown as SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 26, and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 21. SEQ ID NO: 22 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 23; or

vi) the heavy chain variable region comprises the sequences shown in SEQ ID NOs: 24. SEQ ID NO: 28 and SEQ ID NO: 26, and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 27. SEQ ID NO: 22 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in fig. 23. In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region, wherein:

(A) the heavy chain variable region is identical to SEQ ID NO:9 and/or the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 10 sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical;

(B) the heavy chain variable region is identical to SEQ ID NO:11, and/or the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 12 sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; or

(C) The heavy chain variable region is identical to SEQ ID NO:13, and/or the light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 14 sequences are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above, comprising a heavy chain variable region and a light chain variable region as shown below:

(D) the heavy chain variable region sequence is shown as SEQ ID NO 9, and the light chain variable region sequence is shown as SEQ ID NO: 10 is shown in the figure;

(E) the heavy chain variable region sequence is shown as SEQ ID NO:11, and the light chain variable region sequence is shown as SEQ ID NO: 12 is shown in the specification; or

(F) The heavy chain variable region sequence is shown as SEQ ID NO:13, and the light chain variable region sequence is shown as SEQ ID NO: as shown at 14.

In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above, wherein the anti-ANGPTL 3 antibody further comprises a heavy chain constant region and a light chain constant region; in some embodiments, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from the group consisting of human kappa and lambda chain constant regions; in some embodiments, the heavy chain constant region sequence is as set forth in SEQ ID NO. 29 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the SEQ ID NO. 29 sequence and/or the light chain constant region sequence is as set forth in SEQ ID NO. 30 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the SEQ ID NO. 30 sequence. In some embodiments, the anti-ANGPTL 3 antibody or antigen binding fragment thereof, as described above, wherein the anti-ANGPTL 3 antibody comprises a heavy chain and a light chain, wherein,

(G) the heavy chain has the same structure as SEQ ID NO: 31, and/or the light chain is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 32 sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical;

(H) the heavy chain has the same structure as SEQ ID NO: 33, and/or the light chain is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 34 sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical; or

(I) The heavy chain has the same structure as SEQ ID NO: 35, and/or the light chain is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36 sequences are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above, wherein the anti-ANGPTL 3 antibody comprises a heavy chain and a light chain, wherein,

(J) the heavy chain sequence is shown as SEQ ID NO: 31 and the light chain sequence is shown in SEQ ID NO: 32 is shown;

(K) the heavy chain sequence is shown as SEQ ID NO: 33, and a light chain sequence as set forth in SEQ ID NO: 34; or

(L) the heavy chain sequence is shown as SEQ ID NO: 35 and the light chain sequence is shown in SEQ ID NO: shown at 36.

In some embodiments, the present disclosure provides an isolated anti-ANGPTL 3 antibody or antigen-binding fragment thereof that competes for binding to an ANGPTL3 antigen with an aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof. In some embodiments, the ANGPTL3 antigen is human ANGPTL3, mouse ANGPTL3, monkey ANGPTL3, rat ANGPTL 3.

In some embodiments, the antibodies of the disclosure are human antibodies or human-derived antibodies. In some embodiments, the antigen binding fragment of the disclosure is selected from the group consisting of: fab, F (ab ')2, Fab', Fd, Fv, dsFv, scFv, Fab and diabodies. In some embodiments, an anti-ANGPTL 3 antibody or antigen binding fragment thereof of the present disclosure has at least one of the following a-d characteristics:

a. specifically binds to ANGPTL3 protein. In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof binds to human ANGPTL3, mouse ANGPTL3, cynomolgus monkey ANGPTL3, and/or rat ANGPTL 3L with an EC50 of less than 10nM (e.g., less than or equal to 5nM, less than or equal to 3nM, less than or equal to 1nM, less than or equal to 0.8nM, less than or equal to 0.6nM, less than or equal to 0.4nM, less than or equal to 0.3nM, less than or equal to 0.2 nM); in some embodiments, the EC50 is determined by an ELISA method, such as described in test example 1 of the present disclosure; in some embodiments, the EC50 is determined by HTRF methods, such as described in test example 2 of the present disclosure;

b. binds ANGPTL3 protein with high affinity. In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof binds to a human ANGPTL3, mouse ANGPTL3, cynomolgus monkey ANGPTL3, and/or rat ANGPTL3 antigen with a KD value of less than 10E-10M (e.g., less than or equal to 9E-10M, less than or equal to 8E-10M, less than or equal to 7E-10M, less than or equal to 6E-10M, less than or equal to 3E-10M, less than or equal to 2E-10M, less than or equal to 1E-10M, less than or equal to 8E-11M, less than or equal to 6E-11M, less than or equal to 5E-11M); in some embodiments, the KD values are determined by a Biacore method, such as described in test example 4 of the present disclosure;

c. block the inhibiting effect of ANGPTL3 on LPL enzyme. In some embodiments, the anti-ANGPTL 3 antibody or antigen-binding fragment thereof blocks inhibition of LPL enzyme activity by human ANGPTL3, mouse ANGPTL3, cynomolgus monkey ANGPTL3, and/or rat ANGPTL3 with an IC50 value of less than 170nM (e.g., less than or equal to 80nM, less than or equal to 135nM, less than or equal to 80nM, less than or equal to 55 nM). The IC50 may be determined by an enzyme activity assay, such as described in test example 3 of the present disclosure; and/or

d. Has good effect of reducing blood fat. In some embodiments, the lipid lowering effect of an anti-ANGPTL 3 antibody or antigen binding fragment thereof of the present disclosure is as shown in test examples 5, 6, and/or 7. In some embodiments, the disclosure provides a nucleic acid molecule encoding the aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof.

In some embodiments, the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.

In some embodiments, the present disclosure provides a host cell comprising the aforementioned nucleic acid molecule. In some embodiments, the present disclosure provides a host cell obtained by transformation (or transduction, transfection) of the aforementioned vector; the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells. In some embodiments, the host cell does not include any human cells capable of developing into a whole individual, such as human embryonic stem cells, fertilized eggs, germ cells; in some embodiments, the host cell is a eukaryotic cell, more preferably a mammalian cell, including but not limited to CHO, 293, NSO, and a cell in which gene editing can alter glycosylation modification of an antibody or antigen binding fragment thereof, thereby altering ADCC function of the antibody or antigen binding fragment thereof, e.g., knock-out of the genes FUT8 or GnT-III; in some embodiments, the mammalian cell does not comprise a human cell.

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or the aforementioned nucleic acid molecule, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients. In some embodiments, the pharmaceutical composition contains 0.01 to 99% by weight of the aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or the aforementioned nucleic acid molecule, in a unit dose. In some embodiments, the pharmaceutical composition contains 0.1 to 2000mg, more preferably 1 to 1000mg, of the aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or the aforementioned nucleic acid molecule, in a unit dose.

In some embodiments, the disclosure provides a method for immunodetection or assay of ANGPTL3, the method comprising the aforementioned anti-ANGPTL 3 antibody or antigen binding fragment thereof. In another aspect, the disclosure provides a method of detecting the level of ANGPTL3 expression in a sample from a subject in vitro, the method comprising the step of contacting an anti-ANGPTL 3 antibody, or antigen-binding fragment thereof, described in the disclosure with the sample; in some embodiments, the sample is a plasma, serum, whole blood sample from a subject. In some embodiments, the expression level of ANGPTL3 can be detected using any method known to those of skill in the art, including but not limited to: western blot, immunoblot, ELISA, mass spectrometry.

In some embodiments, the disclosure provides a kit comprising the aforementioned anti-ANGPTL 3 antibody or antigen-binding fragment thereof.

In some embodiments, the disclosure provides a method of making an anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above.

In some embodiments, the disclosure provides the use of an anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described above in the preparation of a reagent for the immunodetection of ANGPTL 3.

In some embodiments, the disclosure provides an anti-ANGPTL 3 antibody or antigen-binding fragment thereof as described previously for use in immunodetection or assay of ANGPTL 3;

in some embodiments, the disclosure provides a kit comprising an anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to the foregoing. In some embodiments, the disclosure provides a kit for detecting expression levels of ANGPTL3, comprising an anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to the foregoing. In some embodiments, the kit is used in a method of detecting the expression level of ANGPTL3 described in the present disclosure; in some embodiments, the detection method is any method known to those of skill in the art, including, but not limited to, Western blot, immunoblot, ELISA, mass spectrometry.

In some embodiments, the disclosure provides a method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of an anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a pharmaceutical composition, as described above; in some embodiments, the disease or disorder is an ANGPTL 3-associated disease or disorder; in some embodiments, the disease or disorder is hypercholesterolemia, hyperlipidemia, or an atherosclerotic disease.

In some embodiments, the disclosure provides the use of an anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a pharmaceutical composition as described above, in the manufacture of a medicament for treating a disease or disorder. In some embodiments, the disease or disorder is an ANGPTL 3-associated disease or disorder; in some embodiments, the disease or disorder is hypercholesterolemia, hyperlipidemia, or an atherosclerotic disease.

In some embodiments, the disclosure provides an anti-ANGPTL 3 antibody or antigen-binding fragment thereof, or a nucleic acid molecule, or a pharmaceutical composition, as described previously, as a medicament. In some embodiments, the medicament is for treating an ANGPTL 3-associated disease or disorder; in some embodiments, the disease or disorder is hypercholesterolemia, hyperlipidemia, or an atherosclerotic disease.

Detailed Description

Term (definition)

In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The three letter and one letter codes for amino acids used in the present disclosure are as described in j. biol. chem, 243, p3558 (1968).

An "antibody" as described in the present disclosure refers to an immunoglobulin, a natural intact antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. Depending on the amino acid composition and arrangement of the constant region of an immunoglobulin heavy chain, immunoglobulins can be classified into five classes, otherwise known as the immunoglobulin isotypes, i.e., IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, IgG can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.

The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3.

"antibodies" of the present disclosure include, in addition to full-length antibodies, antigen-binding fragments that bind antigen.

Antibodies of the disclosure include human antibodies.

The term "murine antibody" is in this disclosure a monoclonal antibody directed against human ANGPTL3 prepared according to the knowledge and skill in the art. The test subjects can be injected with the ANGPTL3 antigen at the time of preparation, and the hybridomas expressing antibodies with the desired sequence or functional properties can then be isolated. In a preferred embodiment of the disclosure, the murine anti-ANGPTL 3 antibody or antigen binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or variant thereof.

The term "chimeric antibody" refers to an antibody in which the variable region of a heterologous (e.g., murine) antibody is fused to the constant region of a parent antibody (e.g., human antibody) to reduce the immune response induced by a heterologous antibody. For example, a human-mouse chimeric antibody is created by creating a hybridoma secreting a specific monoclonal antibody of murine origin, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody as required, linking the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the disclosure, the antibody light chain of the anti-ANGPTL 3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the ANGPTL3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof, preferably comprises a human IgG1, IgG2, or IgG4 heavy chain constant region, or an IgG1, IgG2, or IgG4 variant using amino acid mutations (e.g., L234A and/or L235A mutations, and/or S228P mutations).

The term "humanized antibody", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Can overcome the heterogenous reaction induced by the chimeric antibody carrying a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. Germline DNA Sequences of genes such as the human heavy and light chain variable regions are available at the "VBase" human germline sequence database and are found in Kabat, E.A. et al, 1991Sequences of Proteins of Immunological Interest, 5 th edition. To avoid reduced immunogenicity and reduced activity, the human antibody variable region framework sequences may be minimally back-mutated or back-mutated to retain activity. Humanized antibodies of the disclosure also include humanized antibodies after further affinity maturation mutation of the CDRs by yeast display.

The terms "human antibody" and "human antibody" are used interchangeably to mean that one or more variable and constant regions are derived from human immunoglobulin sequences. One preferred way is that all variable and constant regions are derived from human immunoglobulin sequences, i.e., "fully human antibodies" or "fully human antibodies". These antibodies can be prepared by a variety of means, including the construction of natural single-chain phage human antibody libraries by phage display techniques, isolation of B cells from human PBMC, spleen, lymph node tissues, or by immunization of transgenic mice expressing human antibody light and heavy chains, and screening of the resulting antibodies. The human antibodies of the present disclosure also include antibodies that still bind the antigen of interest, obtained by mutation of one or more amino acids on the basis of human antibodies.

The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region in the present disclosure refer to variants of the heavy chain constant region or light chain constant region that have been disclosed in the prior art to be of human origin without altering the structure and function of the antibody variable region, and exemplary variants include variants of the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4 heavy chain constant region with site-directed modification and amino acid substitution, specifically substitution such as YTE mutation known in the prior art, L234A and/or L235A mutation, S228P mutation, and/or mutation to obtain a knob-into-hole structure (such that the antibody heavy chain has a combination of knob-Fc and hole-Fc), which have been confirmed to give the antibody new properties without altering the function of the antibody variable region.

The terms "full-length antibody," "intact antibody," "complete antibody," and "whole antibody" are used interchangeably herein to refer to a substantially intact form of an antibody, as distinguished from antigen-binding fragments defined below. The term particularly refers to antibodies in which the light and heavy chains comprise constant regions. The disclosure "antibody" includes "full-length antibodies" and antigen-binding fragments thereof.

In some embodiments, the full-length antibodies of the disclosure include full-length antibodies formed by a light chain variable region linked to a light chain constant region and a heavy chain variable region linked to a heavy chain constant region. Those skilled in the art can select different antibody-derived light chain constant regions and heavy chain constant regions, such as human antibody-derived light chain constant regions and heavy chain constant regions, according to actual needs.

The term "antigen-binding fragment" or "functional fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., ANGPTL 3). It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) f (ab') ₂ A fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (ii) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) dsFv, a stable antigen-binding fragment formed by interchain disulfide bonds from VH and VL; (vi) diabodies, bispecific antibodies and multispecific antibodies comprising fragments of scFv, dsFv, Fab and the like. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, enabling their generation of a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science242: 423-; and Huston et al (1988) Proc. Natl. Acad. Sci USA85: 5879-. Such single chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

The Fab can be obtained as an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity by treating an IgG antibody molecule with papain, a protease which cleaves the amino acid residue at position 224 of the H chain, in which about half of the N-terminal side of the H chain and the entire L chain are bound together by a disulfide bond.

F (ab')2 an antibody fragment having a molecular weight of about 100,000 and antigen binding activity and comprising two Fab regions linked at the hinge position can be obtained by pepsin digestion of the lower part of the two disulfide bonds in the IgG hinge region.

Fab 'is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity, which can be obtained by cleaving the disulfide bond of the hinge region of the above-mentioned F (ab') 2. Fab's of the present disclosure can be produced by treating F (ab')2 of the present disclosure that specifically recognizes ANGPTL3 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.

In addition, the Fab ' may be produced by inserting DNA encoding the Fab ' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab '.

The term "single chain antibody", "single chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior artThe linker consists of a repeated GGGGS amino acid sequence or a variant thereof, for example using a 1-4 repeated variant (Holliger et al (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al (1995), Protein Eng.8: 725-.

Diabodies are antibody fragments in which the scFv or Fab is dimerized, and are antibody fragments having bivalent antigen binding activity. In the divalent antigen binding activity, the two antigens may be the same or different.

Bispecific and multispecific antibodies refer to antibodies that bind two or more antigens or antigenic determinants simultaneously, comprising an scFv or Fab fragment that binds ANGPTL 3.

Diabodies of the disclosure can be produced by the following steps: obtaining cdnas encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human ANGPTL3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof, constructing DNA encoding scFv such that the amino acid sequence of the peptide linker is 8 residues or less in length, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.

The dsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue via a disulfide bond between cysteine residues. The amino acid residue substituted with a cysteine residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).

A full-length antibody or antigen-binding fragment of the disclosure can be produced by: obtaining cDNA encoding the antibody of the present disclosure that specifically recognizes human ANGPTL3 and binds to the amino acid sequence of the extracellular region or a three-dimensional structure thereof, constructing DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryote or a eukaryote to express dsFv.

The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide as compared to the original protein or polypeptide, including insertions, deletions or substitutions of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of that variable domain. It is essentially a variable domain without CDRs.

The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the 6 hypervariable regions within the variable domain of an antibody which primarily contributes to antigen binding. Typically, there are three CDRs per heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs per light chain variable region (LCDR1, LCDR2, LCDR 3). The amino acid sequence boundaries of the CDRs may be determined using any of a variety of well-known protocols, including "Kabat" numbering convention (see Kabat et Al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering convention (see Al-Lazikani et Al, (1997) JMB 273: 927-948) and ImmunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunological, 7, 132-136 (1999); Lefranc, M.P., et Al, Dev.Comp., 27, 55-77 (Im et Al).

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on the ANGPTL3 molecule). Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, e.g., epipope Mapping Protocols in Methods in Molecular B biology, volume 66, g.e. morris, Ed. (1996).

The terms "specifically binds", "selectively binds" and "specifically binds", "is capable of specifically binding" refer to the antibody pair pre-bindingBinding of an epitope on a previously determined antigen. Typically, the antibody is administered at a rate of about less than 10 ^-8 M, e.g. less than about 10 ^-9 M、10 ^-10 M、10 ^-11 M、10 ^-12 M or less affinity (KD) binding.

The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, antibodies of the disclosure are administered at less than about 10 ^-7 M, e.g. less than about 10 ^-8 M or 10 ^-9 The dissociation equilibrium constant (KD) of M binds to ANGPTL3, e.g., the affinity of an antibody to a cell surface antigen in the present disclosure is determined by FACS methods for KD values; in other embodiments, the affinity KD values of the antibody to the antigen are determined using the Biacore method; in other embodiments, binding of the antibody to the antigen is determined using an ELISA method.

The term "compete" when used in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins, as determined by the following assay: in the assay, the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., an ANGPTL3 antigen or fragment thereof). Numerous types of competitive binding assays are available for determining whether an antigen binding protein competes with another, such as: solid phase direct or indirect Radioimmunoassays (RIA), solid phase direct or indirect Enzyme Immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al, 1983, methods in Enzymology 9: 242-; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al, 1986, J.Immunol.137: 3614-), solid phase direct labeling assay, solid phase direct labeling sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); direct labeling of RIA with a solid phase of I-125 label (see, e.g., Morel et al, 1988, mol. Immunol.25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al, 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al, 1990, Scand. J. Immunol.32: 77-82). Typically, the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being measured. Typically the antigen binding protein to be detected is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: an antigen binding protein that binds to the same epitope as a reference antigen binding protein; and an antigen binding protein that binds a contiguous epitope sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically hinder binding from occurring. Additional details regarding methods for determining competitive binding are provided in the examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., decrease) the specific binding of at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of a reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term "nucleic acid molecule" as used herein refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to amino acid residues in a second sequence, when the amino acid sequences are aligned and gaps are introduced, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. For purposes of determining percent amino acid sequence identity, alignments can be accomplished in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in a host cell into which they have been introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of a host cell upon introduction into the host cell so as to be replicated along with the host genome (e.g., non-episomal mammalian vectors).

Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as the Cold spring harbor antibody protocols, chapters 5-8 and 15. For example, mice can be immunized with human ANGPTL3 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods. The antigen binding fragment may also be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to incorporate one or more FR regions of human origin in a CDR region of non-human origin. Human FR germline sequences can be obtained from the website of ImmunoGeneTiCs (IMGT) or from the immunoglobulin journal, 2001ISBN012441351, by aligning the IMGT human antibody variable region germline gene database with the MOE software.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria susceptible to transformation include members of the enterobacteriaceae family (enterobacteriaceae), such as strains of Escherichia coli (Escherichia coli) or Salmonella (Salmonella); bacillaceae (Bacillus) such as Bacillus subtilis; pneumococcus (Pneumococcus); streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae). Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (chinese hamster ovary cell line), 293 cells and NS0 cells.

Engineered antibodies or antigen-binding fragments of the disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human ANGPTL 3. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified by conventional techniques. For example, purification is carried out using an A or G Sepharose FF column containing a buffer adjusted. Non-specifically bound fractions are washed away. And eluting the bound antibody by using a pH gradient method, detecting the antibody fragment by using SDS-PAGE, and collecting. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering a therapeutic agent, e.g., a composition comprising any one of the binding compounds of the present disclosure, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, to induce regression of such symptoms or to inhibit development of such symptoms to any clinically useful degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of each target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.

"conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity. Exemplary conservative substitutions are set forth in the following table "exemplary amino acid conservative substitutions".

TABLE 1 exemplary amino acid conservative substitutions

Original residue	Conservative substitutions
Ala(A)	Gly；Ser
Arg(R)	Lys；His
Asn(N)	Gln；His；Asp
Asp(D)	Glu；Asn
Cys(C)	Ser；Ala；Val
Gln(Q)	Asn；Glu
Glu(E)	Asp；Gln
Gly(G)	Ala
His(H)	Asn；Gln
Ile(I)	Leu；Val
Leu(L)	Ile；Val
Lys(K)	Arg；His
Met(M)	Leu；Ile；Tyr
Phe(F)	Tyr；Met；Leu
Pro(P)	Ala
Ser(S)	Thr
Thr(T)	Ser
Trp(W)	Tyr；Phe
Tyr(Y)	Trp；Phe
Val(V)	Ile；Leu

An "effective amount" or "effective dose" refers to the amount of a drug, compound or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, lessening the severity, or delaying the onset of the condition, including biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes exhibited during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of or ameliorating one or more symptoms of various target antigen-associated disorders of the disclosure, reducing the dosage of other agents required to treat the disorder, enhancing the therapeutic efficacy of another agent, and/or delaying the progression of a target antigen-associated disorder of the disclosure in a patient.

"exogenous" refers to a substance produced outside an organism, cell or human body as the case may be. "endogenous" refers to a substance produced in a cell, organism, or human body as the case may be.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clearly visible from the context.

As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which minute amounts of a particular portion of nucleic acid, RNA, and/or DNA are amplified as described, for example, in U.S. patent No. 4,683,195. In general, it is desirable to obtain sequence information from the ends of or beyond the target region so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotide of the 2 primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al (1987) Cold Spring Harbor Symp. Ouant. biol.51: 263; erlich editors, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or generate specific portions of the nucleic acid.

"isolated" refers to the purified state, and in this case means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to refer to the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivery of the antibody or antigen binding fragment. The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (e.g., antioxidant, antibacterial or antifungal agent), sweetener, absorption retarder, wetting agent, emulsifier, buffer, or the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

Furthermore, the disclosure includes agents for treating diseases associated with antigen-positive cells of interest (e.g., ANGPTL3) comprising as an active ingredient an anti-ANGPTL 3 antibody or antigen-binding fragment thereof of the disclosure.

The disease associated with ANGPTL3 in the present disclosure is not limited as long as it is a disease associated with ANGPTL3, e.g., a therapeutic response induced by a molecule of the present disclosure can be by binding to human ANGPTL3, and then blocking the binding of ANGPTL3 to its ligand. Thus, the molecules of the present disclosure are very useful in humans suffering from hypercholesterolemia, hyperlipidemia, or atherosclerotic disease, among others, when in preparations and formulations suitable for therapeutic use.

Furthermore, the present disclosure relates to a method for immunodetection or assay of a target antigen (e.g., ANGPTL3), a reagent for immunodetection or assay of a target antigen (e.g., ANGPTL3), a method for immunodetection or assay of a cell expressing a target antigen (e.g., ANGPTL3), and a diagnostic agent for diagnosis of a disease associated with a target antigen (e.g., ANGPTL3) -positive cell, which comprises, as an active ingredient, an antibody or antibody fragment of the present disclosure that specifically recognizes a target antigen (e.g., human ANGPTL3) and binds to an amino acid sequence of an extracellular region or a three-dimensional structure thereof.

In the present disclosure, the method for detecting or determining the amount of an antigen of interest (e.g., ANGPTL3) can be any known method. For example, it includes immunodetection or assay methods.

The immunoassay or measuring method is a method for detecting or measuring the amount of an antibody or the amount of an antigen using a labeled antigen or antibody. Examples of the immunological detection or measurement method include a radioactive substance-labeled immune antibody method (RIA), an enzyme immunoassay (EIA or ELISA), a Fluorescence Immunoassay (FIA), a luminescence immunoassay, a western immunoblotting method, a physicochemical method, and the like.

The above-described diseases associated with ANGPTL 3-positive cells can be diagnosed by detecting or assaying cells expressing ANGPTL3 with an antibody or antibody fragment of the disclosure.

For detecting cells expressing the polypeptide, a known immunoassay method can be used, and immunoprecipitation, fluorescent cell staining, immunohistological staining, or the like is preferably used. In addition, a fluorescent antibody staining method using FMAT8100HTS system (Applied Biosystem) or the like can be used.

In the present disclosure, the living sample for detecting or measuring a target antigen (e.g., ANGPTL3) is not particularly limited as long as it has a possibility of containing cells expressing the target antigen (e.g., ANGPTL3), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may further contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction, depending on the desired diagnostic method. Reagents for performing antigen-antibody reactions include buffers, salts, and the like. The reagent for detection includes reagents generally used in immunodetection or assay methods, such as a labeled secondary antibody recognizing the monoclonal antibody, an antibody fragment thereof or a binding substance thereof, a substrate corresponding to the label, and the like.

The details of one or more embodiments of the invention are set forth in the description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in the specification are herein incorporated by reference. The following examples are set forth in order to more fully illustrate the preferred embodiments of the present invention. These examples should not be construed in any way to limit the scope of the invention, which is defined in the claims.

Drawings

FIG. 1: effect of P8BG antibody on SD (Sprague Dawley, sporeleger dorey) rat serum triglycerides;

FIG. 2: effect of P3G antibody on SD rat serum triglycerides;

FIG. 3: effect of anti-ANGPTL 3 antibodies on HFD (High-fat-diet) mouse plasma triglycerides;

FIG. 4: effect of anti-ANGPTL 3 antibody on HFD mouse plasma Low Density Lipoprotein Cholesterol (Low-sensitivity Lipoprotein Cholesterol, LDL-C);

FIG. 5: effect of anti-ANGPTL 3 antibodies on HFD mouse plasma total cholesterol;

FIG. 6: effect of anti-ANGPTL 3 antibodies on APOE mouse (apolipoprotein E, APOE) gene knockout mice) plasma triglycerides;

FIG. 7: effect of anti-ANGPTL 3 antibodies on APOE mouse plasma High density lipoprotein cholesterol (HDL-C);

FIG. 8: results of in vivo pharmacokinetic experiments of anti-ANGPTL 3 antibodies in mice;

FIG. 9: results of pharmacokinetic experiments in cynomolgus monkeys of anti-ANGPTL 3 antibody.

Detailed Description

The disclosure is further described below in conjunction with the following examples, which, however, do not limit the scope of the disclosure.

Experimental procedures in examples or test examples of the present disclosure, in which specific conditions are not specified, are generally performed under conventional conditions or conditions recommended by manufacturers of raw materials or commercial products. See Sambrook et al, molecular cloning, A laboratory Manual, Cold spring harbor laboratory; contemporary molecular biology methods, Ausubel et al, Greene publishing Association, Wiley Interscience, NY. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Examples

Example 1 ANGPTL3 antigen design and expression

Human ANGPTL3 protein (Uniprot No. Q9Y5C1), mouse ANGPTL3 protein (Uniprot No. Q9R182), cynomolgus monkey ANGPTL3 protein (Uniprot No. A0A2K5UDC5) and rat ANGPTL3 protein (Uniprot No. F7FHP0) are used as templates of the ANGPTL3, the antigen and the protein for detection related to the disclosure are designed, different labels are fused on the basis of the ANGPTL3 protein, and are respectively cloned on pTT5 vector (Biovector, Cat #:102762), and the antigen and the protein for detection are obtained by transient transfection expression in 293 cells and purification.

1. Human full-length ANGPTL 3: human-ANGPTL 3(17-460) -His, useful for immunization and detection, having the following amino acid sequence:

note that: the signal peptide sequence is single underlined, the His sequence is italicized, and the amino acids 17 to 460 of the full-length human ANGPTL3 protein are bold.

2. Human ANGPTL3 extracellular region: human ANGPTL3(17-170) -Flag-His, which can be used for immunization and detection, and has the following amino acid sequence:

note that: the signal peptide is single underlined, the linker peptide is double underlined, Flag and His are italicized, and amino acids 17 to 170 of the full-length human ANGPTL3 protein are bolded.

3. Fusion protein of the extracellular region of human ANGPTL3 (human ANGPTL3(17-170)) and the mouse IgG2aFc fragment (abbreviation: mFc): human ANGPTL3(17-170) -mFc which can be used for detection and has the following amino acid sequence:

note that: the signal peptide is underlined, the mouse IgG2aFc is italicized, and the amino acids 17 to 170 of the human ANGPTL3 full-length protein are bolded.

4. Fusion protein of the extracellular region of human ANGPTL3 (human ANGPTL3(17-220)) and the mouse IgG2aFc fragment: human ANGPTL3(17-220) -mFc for immunization and detection, the amino acid sequence of which is as follows:

and (3) annotation: the signal peptide is underlined, the mouse IgG2aFc is italicized, and the amino acids 17 to 220 of the human ANGPTL3 full-length protein are bolded.

5. Mouse full length ANGPTL 3: mouse ANGPTL3(17-455) -His, for immunization and detection, has the following amino acid sequence:

note that: the signal peptide is underlined, the His sequence is italicized, and the amino acids 17 to 455 of the full-length mouse ANGPTL3 protein are in bold.

6. Fusion protein of mouse ANGPTL3 extracellular region (mouse-ANGPTL3(17-220)) and mouse IgG2aFc segment: mouse ANGPTL3(17-220) -mFc for immunization, the amino acid sequence of which is as follows:

note that: the signal peptide is underlined, the mouse IgG2aFc is italicized, and the amino acids 17 to 220 of the full-length mouse ANGPTL3 protein are in bold.

7. Cynomolgus full-length ANGPTL 3: cynomolgus monkey ANGPTL3(17-460) -His, for detection, has the following amino acid sequence:

note that: the signal peptide is underlined, the His sequence is in italics, and the amino acids 17 to 460 of the cynomolgus ANGPTL3 full-length protein are in bold.

8. Rat full-length ANGPTL 3: rat ANGPTL3(17-455) -His, for detection, whose amino acid sequence is as follows:

note that: the signal peptide is the underlined, the His sequence is the italicized part, and the amino acids 17 to 455 of the full-length rat ANGPTL3 protein are in bold.

Example 2 purification of ANGPTL3 antigenic protein, hybridoma antibody, recombinant antibody

Purification of His-tagged recombinant protein or Flag-His-tagged recombinant protein

The cell expression samples were centrifuged at high speed to remove cells and insoluble impurities, the supernatant was collected and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with a PBS solution containing 5mM imidazole, and washed 2-5 column volumes. Cell supernatants were applied to the column. The column was washed with a PBS solution containing 5mM imidazole until the a280 reading dropped to baseline. The column was then washed with PBS +10mM imidazole to remove non-specifically bound contaminating proteins and the flow-through was collected. The target protein was eluted with 300mM imidazole in PBS and the peak was collected.

And further purifying the collected eluent by a Superdex gel filtration column, and collecting the eluent by tubes. The obtained sample is identified to be correct through SDS-PAGE, a peptide diagram and LC-MS and then is subpackaged for standby. Obtaining the recombinant protein with His label or the recombinant protein with Flag-His label.

Purification of hybridoma antibodies or recombinant proteins with mFc tags

The sample was centrifuged at high speed to remove cells and insoluble impurities, leaving the supernatant. The Protein A affinity column was equilibrated with 1 XPBS and washed 2-5 times the column volume. The centrifuged cell expression supernatant sample was applied to the column. The column was washed with 1 × PBS until the a280 reading dropped to baseline. The column was washed with 1 × PBS. The target protein was eluted with 0.1M acetic acid (pH3.5-4.0) and collected, immediately after which the pH of the eluted protein was adjusted to neutrality with 1M Tris-HCl (pH 7.0), and finally dialyzed or concentrated into 1 XPBS buffer. Collecting samples, identifying the samples correctly through electrophoresis, peptide mapping and LC-MS, and subpackaging for later use. Obtaining the recombinant protein or hybridoma antibody with the mFc label.

Purification of antibody and Fc-tagged recombinant protein

The sample was centrifuged at high speed to remove cells and insoluble impurities, leaving the supernatant. The Protein G affinity column was equilibrated with 1 XPBS and washed 2-5 times the column volume. The centrifuged cell expression supernatant sample was applied to the column. The column was washed with 1 × PBS until the a280 reading dropped to baseline. The column was washed with 1 × PBS. The target protein was eluted with acetic acid (pH 3.0) and collected, immediately after which the pH of the eluted protein was adjusted to neutrality with 1M Tris-HCl (pH 7.0), and finally dialyzed or concentrated for exchange into 1 XPBS buffer. Collecting samples, identifying the samples correctly through electrophoresis, peptide diagram and LC-MS, and subpackaging for later use. Obtaining the antibody or the recombinant protein with the hFc label.

Example 3 preparation of anti-ANGPTL 3 antibodies

Screening of anti-ANGPTL 3 humanized antibody by phage library

Packaging and concentrating the humanized phage single-chain antibody library, and packaging the phage library (10) ¹² ～10 ¹³ Pfu) was suspended in 1ml of 2% MPBS (PBS containing 2% skim milk powder) and 100. mu.l was added

M-280 streptavidin (Invitrogen Cat No.11206D) was placed on a turntable and turned over repeatedly, and blocked at room temperature for 1 hour. The tube was placed on a magnetic rack for 2 minutes, the Dynabeads were removed, and the phage library was transferred to a new tube. 2. mu.g/ml biotin-labeled human ANGPTL3(17-460) -His was added to the phage pool after blocking, and placed on a rotary stage and repeatedly tumbled for 1 hour. All in oneThen 100. mu.l Dynabeads were suspended in 1ml of 2% MPBS, placed on a turntable and turned over repeatedly, and sealed at room temperature for 1 hour. Place the tube on a magnetic stand for 2 minutes and aspirate off the blocking solution. The blocked Dynabeads were added to the phage pool and human ANGPTL3(17-460) -His mixture and placed on a rotating platform and repeatedly tumbled for 15 minutes. The tube was placed on a magnetic stand for 2 minutes and the mixture was aspirated. Dynabeads were washed 10 times with 1ml of PBST (PBS containing 0.1% Tween-20), 0.5ml of 1MG/ml trypsin (Sigma Cat No. T1426-250MG) was added, the mixture was placed on a rotary table and incubated for 15 minutes with repeated inversion, elution was performed, the eluted phage were infected with E.coli TG1 and plated, and single clones were randomly picked for phage ELISA.

The clones were inoculated into a 96-well deep-well plate (Nunc Cat No.260251) and cultured at 37 ℃ for 16 to 18 hours. A small amount of the suspension was inoculated into another 96-well deep-well plate until the OD600 reached about 0.5, and M13K07 helper phage (NEB Cat No. N0315S) was added for packaging. And (4) centrifuging at 4000g for 10 minutes to remove bacteria, and sucking culture solution to carry out ANGPTL3 combined ELISA detection. The positive clone strains are frozen and preserved and sent to a sequencing company for sequencing. Wherein, the amino acid sequences corresponding to the positive clones P3 and P8 are as follows:

p3 heavy chain variable region sequence:

p3 light chain variable region sequence:

p8 heavy chain variable region sequence:

p8 light chain variable region sequence:

remarking: the underlined parts of the sequence are the CDR sequences determined according to the Kabat numbering system, and the FR sequences are italicized.

Second, mutation of anti-ANGPTL 3 antibody

Based on the three-dimensional structure of the P8 antibody, the 55 th amino acid residue (natural sequence number) in the P8 heavy chain variable region is mutated from D to E, and the 34 th amino acid residue (natural sequence number) in the P8 light chain variable region is mutated from G to V, so as to obtain a novel antibody molecule P8B, wherein the amino acid sequence of P8B is as follows:

(> P8B heavy chain variable region sequence:

P8B light chain variable region sequence:

TABLE 2 CDR sequences of anti-ANGPTL 3 antibody molecules

Construction and expression of anti-ANGPTL 3 antibody

Designing a primer PCR to build an antibody VH/VK gene segment, and then carrying out homologous recombination with an expression vector pHr (with a signal peptide and a constant region gene (CH1-FC/CL) segment) to construct an antibody full-length expression vector VH-CH 1-FC-pHr/VK-CL-pHr. The heavy chain constant region of the antibody may be selected from the constant regions of human IgG1, IgG2, IgG4 and variants thereof, and the light chain constant region may be selected from the light chain constant regions of human kappa, lambda chains or variants thereof. Illustratively, the antibody heavy chain constant region is selected from the group consisting of SEQ ID NOs: 29, light chain constant region selected from the group consisting of human IgG4-YTE variants having the sequence shown in SEQ ID NO:30, constant region of human kappa chain.

Human IgG4-YTE variant heavy chain constant region sequence:

human kappa chain light chain constant region sequence:

illustratively, the light/heavy chain constant regions described above in combination with the variable regions of the aforementioned P3, P8, P8B form the complete anti-ANGPTL 3 antibody: P3G, P8G, P8BG, the light/heavy chain sequence of the antibody is as follows:

(> P3G heavy chain sequence:

(> P3G light chain sequence:

(> P8G heavy chain sequence:

(> P8G light chain sequence:

(> P8BG heavy chain sequence:

(> P8BG light chain sequence:

the following biochemical test method to verify the activity of the antibodies of the disclosure

Test example 1 ELISA assay for anti-ANGPTL 3 antibody binding to ANGPTL3 protein

An anti-ANGPTL 3 antibody inhibits or interferes with at least one activity of an ANGPTL3 protein by binding to the ANGPTL3 protein. The binding activity of anti-ANGPTL 3 antibodies to ANGPTL3 antigen was tested in an ELISA assay. The ANGPTL3 protein (human ANGPTL3(17-170) -mFc) was immobilized in a 96-well microplate by binding to goat anti-mouse IgG coated on the microplate, and the binding activity of the antibody and ANGPTL3 was judged according to the intensity of the signal after the antibody was added. Meanwhile, the HIS-tagged ANGPTL3 protein (mouse ANGPTL3(17-455) -His, cynomolgus monkey ANGPTL3(17-460) -His, rat ANGPTL3(17-455) -His) labeled with a biotin labeling kit (east Kernel chemical, LK03) was immobilized in a 96-well microplate by binding to streptavidin coated in the microplate, and the binding activity of the antibody and ANGPTL3 was judged according to the intensity of the signal after the antibody was added.

Goat anti-mouse IgG (Sigma, M3534-1ML) was diluted to a concentration of 2. mu.g/ML with PBS (Shanghai culture, B320) buffer, pH7.4, added to a 96-well plate at a volume of 50. mu.l/well (Corning, CLS3590-100EA), and left in an incubator at 37 ℃ for 3 hours or at 4 ℃ overnight (16-18 hours). After discarding the liquid, 250. mu.l/well of 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the cells were incubated at 37 ℃ for 3 hours or left overnight (16-18 hours) at 4 ℃ for blocking. After blocking was complete, the blocking solution was discarded and the plates were washed 3 times with PBST buffer (pH7.4PBS containing 0.05% Tween-20) and then diluted to 0.5. mu.g/ml human ANGPTL3(17-170) -mFc (sequence as SEQ ID NO: 3) by adding 50. mu.l/well of a sample diluent (pH7.4PBS containing 1% BSA) and incubated for 2 hours at 37 ℃. After the incubation is finished, reaction liquid in the enzyme label plate is discarded, the plate is washed by PBST for 3 times, 50 mu l/hole of antibodies to be detected with different concentrations diluted by sample diluent are added, and the mixture is placed in an incubator at 37 ℃ for incubation for 2 hours. After incubation, the plate was washed 3 times with PBST, and 50. mu.l/well of goat anti-human secondary antibody (Jackson Immuno Research, 109-. After washing the plate 3 times with PBST, 50. mu.l/well TMB chromogenic substrate (KPL,52-00-03) was added, incubated at room temperature for 2-5 minutes (min), and 50. mu.l/well 1MH was added ₂ SO ₄ The reaction was stopped, absorbance was read at 450nm using a VersaMax microplate reader, and EC50 values for the binding of anti-ANGPTL 3 antibody to ANGPTL3 antigen protein were calculated. The results are shown in Table 3.

Streptavidin (Sigma, S4762-5MG) was diluted to a concentration of 3. mu.g/ml with PBS (Shanghai culture, B320) buffer, pH7.4, added to a 96-well plate at a volume of 50. mu.l/well (Corning, CLS3590-100EA), and left in an incubator at 37 ℃ for 3 hours or at 4 ℃ overnight (16-18 hours). After discarding the liquid, 250. mu.l/well of 5% skim milk (BD, 232100) blocking solution diluted with PBS was added, and the cells were incubated at 37 ℃ for 3 hours or left overnight (16-18 hours) at 4 ℃ for blocking. After blocking was complete, the blocking solution was discarded, and after washing the plate 3 times with PBST buffer (pH7.4PBS containing 0.05% Tween-20), 50. mu.l/well of a sample diluent (pH7.4PBS containing 0.05% Tween-20) was added1% BSA) to 0.5 μ g/ml biotin-labeled mouse ANGPTL3(17-455) -His (sequence shown in SEQ ID NO: 5) and cynomolgus monkey ANGPTL3(17-460) -His (SEQ ID NO: 7) rat ANGPTL3(17-455) -His (sequence shown in SEQ ID NO: 8) the cells were incubated in an incubator at 37 ℃ for 2 hours. After the incubation is finished, reaction liquid in the enzyme label plate is discarded, the plate is washed by PBST for 3 times, 50 mu l/hole of antibodies to be detected with different concentrations diluted by sample diluent are added, and the mixture is placed in an incubator at 37 ℃ for incubation for 2 hours. After incubation, the plates were washed 3 times with PBST, 50. mu.l/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, 115-. After washing the plate 3 times with PBST, 50. mu.l/well TMB chromogenic substrate (KPL,52-00-03) was added, incubated at room temperature for 2-5 minutes (min), and 50. mu.l/well 1MH was added ₂ SO ₄ The reaction was stopped, absorbance was read at 450nm using a VersaMax microplate reader, and EC50 values for the binding of anti-ANGPTL 3 antibody to ANGPTL3 antigen protein were calculated. The results of the experiments are shown in table 3 and show that the anti-ANGPTL 3 antibodies of the present disclosure are capable of binding to ANGPTL3 proteins of different species.

TABLE 3 binding of anti-ANGPTL 3 antibodies to ANGPTL3 protein of different species ELISA assay

Test example 2 HTRF binding experiment of anti-ANGPTL 3 antibody to ANGPTL3 antigen protein

HTRF experiments were also used to detect the binding activity of anti-ANGPTL 3 antibodies to antigens. HIS-tagged ANGPTL3 (human ANGPTL3(17-460) -His, mouse ANGPTL3(17-455) -His, cynomolgus monkey ANGPTL3(17-460) -His, rat ANGPTL3(17-455) -His) labeled with biotin labeling kit (Donglian chemistry, LK03) was conjugated with HTRF reagent Streptavidin-Tb cryptata (Cisbio, 610SATLA), and after antibody addition, antibody was conjugated with HTRF reagent Pab anti-mouse IgG-XL665(Cisbio, 61PAMXLA) and angiopoietin-like protein 3, respectively, and the magnitude of signal was used to determine the binding activity of antibody and angiopoietin-like protein 3.

Mu.g/ml biotin-labeled human ANGPTL3(17-460) -His (sequence shown as SEQ ID NO: 1), mouse ANGPTL3(17-455) -His (sequence shown as SEQ ID NO: 5), cynomolgus monkey ANGPTL3(17-460) -His (sequence shown as SEQ ID NO: 7), rat ANGPTL3(17-455) -His (sequence shown as SEQ ID NO: 8), 5. mu.l/well were added to the well plate (PerkinElmer, optite-384), then 5. mu.l/well of the HTRF reagent Streptavidin-Tb cryptata and Pab anti-mouse IgG-XL mixed solution 665 were added, finally 10. mu.l/well of the different concentrations of the test antibody diluted with the sample dilution were added and placed at 25 ℃ for 1 hour. Binding EC50 values of anti-ANGPTL 3 antibody to angiopoietin-like protein 3 were calculated by reading values with PHERAstar FS (BMG LabTECH) in HTRF module. The experimental results are shown in table 4, and the implementation results show that the antibody disclosed by the invention has good binding activity to the ANGPTL3 antigen of human, mouse, cynomolgus monkey, rat and other species.

TABLE 4 HTRF binding experiments of anti-ANGPTL 3 antibodies to ANGPTL3 protein

Test example 3 enzyme Activity test for anti-ANGPTL 3 antibody to inhibit LPL

LPL enzyme activity detection experiments are used for detecting the activity of anti-ANGPTL 3 antibody blocking the inhibition effect of ANGPTL3 protein on LPL enzyme.

Bovine LPL (Sigma, L2254-5KU) was diluted with dilution buffer (pH7.4PBS +2mg/mL BSA) to 12.5 units, 25. mu.l/well was added to a 96-well plate (Corning, 3603), then 25. mu.l/well of the antibody to be tested diluted with the sample dilution was added at different concentrations, 25. mu.l/well of ANGPTL3 protein (human ANGPTL3(17-170) -Flag-His (sequence as SEQ ID NO: 2), mouse ANGPTL3(17-455) -His (sequence as SEQ ID NO: 5), cynomolgus ANGPTL3(17-460) -His (sequence as SEQ ID NO: 7), rat ANGPTL3(17-455) -His (sequence as SEQ ID NO: 8) at a concentration of 27.6. mu.g/well was added, 96 was shaken on a shaker, mixed well at 37 ℃ in an incubator for 30min, and finally 25. mu.l/well of Sigma substrate (20. mu.M diluted with the dilution buffer), 30058-10MG-F), shaking the 96-well plate on a shaker, and incubating at 37 deg.C for 30 min. Read in the Flexstation3 microplate reader at Ex535/Em 612. The concentration and corresponding fluorescence data were processed using Graphpad Prism5 software and the IC50 was calculated. The experimental results are shown in table 5, and the implementation results show that the antibodies of the present disclosure have good LPL enzyme inhibition effect.

TABLE 5 Activity of anti-ANGPTL 3 antibodies blocking inhibition of LPL by ANGPTL3 of different species

Test example 4 Biacore detection experiment of anti-ANGPTL 3 antibody and ANGPTL3 protein

Biacore was used to determine the affinity of the anti-ANGPTL 3 antibody to be tested for human, monkey, rat and mouse ANGPTL 3. The method comprises the following steps:

a certain amount of antibodies to be detected are respectively subjected to affinity capture by a Protein A biosensing chip (Cat. #29127556, GE), and then a certain concentration of human, monkey, rat and mouse ANGPTL3 antigens (human-ANGPTL 3(R & D, 3829-AN), mouse ANGPTL3(17-455) -His (sequence is shown as SEQ ID NO: 5), cynomolgus monkey ANGPTL3(17-460) -His (sequence is shown as SEQ ID NO: 7), rat ANGPTL3(17-455) -His (sequence is shown as SEQ ID NO: 8)) are flowed on the surface of the chip. The reaction signal was detected in real time using a Biacore T200 instrument to obtain binding and dissociation curves. After each cycle of dissociation was completed, the biochip was washed clean and regenerated using glycine-hydrochloric acid regeneration solution (Cat. # BR-1003-54, GE) of ph 1.5. The assay run buffer was 1 × HBS-EP buffer (Cat. # BR-1001-88, GE). The data from the experiments were fitted with the (1:1) Langmuir model using GE Biacore T200 evaluation software version 3.0 to yield affinity values. The results are shown in table 6 and demonstrate that the anti-ANGPTL 3 antibodies of the disclosure bind to the ANGPTL3 antigen with high affinity in humans, cynomolgus monkeys, rats, and mice.

TABLE 6 Biacore assay results for anti-ANGPTL 3 antibodies and ANGPTL3 protein

Test example 5 evaluation of in vivo efficacy of anti-ANGPTL 3 antibody in rats

First, P8BG antibody in vivo lipid-lowering experiment in rat

The experimental SD rats were subjected to laboratory environment-adaptive feeding for 1 week (4-week-old SD rats, SPF, about 100g, male, available from Shanghai Si Ricker laboratory animal, Ltd. Certification No. 20170005013017, SCXK 2017-. 7 days before the experiment, the animals are fasted for 4h, then blood is collected in the orbit, and the blood is centrifuged at 3500RPM for 15min to obtain serum. Serum lipid concentrations (triglycerides) were determined using the ADVIA 2400 chemical system (Siemens). Rats were divided into 6 groups by triglyceride split (non-related homo-type human IgG protein (hIgG, the same below) as a negative control group, einacuumab (sequence structure see WHO Drug Information, vol.29, einacuumab sequence in No.3,2015) 3.5mpk group, einacuumab 7mpk group, p8bg3.5mpk group, p8bg1.75mpk group, P8BG7mpk group), 6 rats per group were administered once by subcutaneous injection, and each group of rats was blood-collected after fasting for 4 hours on

days

1, 5, 9, 13, and 16 after injection, and serum lipid concentration (triglyceride) was measured using ADVIA chemical system (Siemens 2400). The experimental data are expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism5 software, and statistically analyzed using TTEST.

As shown in fig. 1 and table 7, compared with the negative control group, after 1 day of administration, triglyceride was decreased in each treatment group, and statistical differences were observed in the groups other than the einacumab3.5 mpk group, and during the experiment, triglyceride was decreased by 83.7% at the maximum in the P8BG7mpk group, 81.8% at the maximum in the P8bg3.5mpk group, and 68.7% at the maximum in the P8BG1.75mpk group; after 5 days of administration, triglyceride reduction of the P8BG3.5mpk group, the P8BG1.75mpk group and the P8BG7mpk group still has statistical difference, while the Eviacimab3.5mpk group and the Eviacimab 7mpk group have no statistical difference; there was still a statistical difference between the P8BG7mpk and P8BG3.5mpk groups after 9 days of dosing. This indicates that P8BG was more effective and longer lasting than evocumab in triglyceride reduction at the same dose. And the animals of each group were normal in weight during the experiment.

TABLE 7 Effect of P8BG antibody on rat serum triglycerides

Remarking: max Inh% represents the maximum percentage of lipid lowering relative to the negative control group TG, mpk represents mg/kg,

second, P3G antibody in vivo lipid-lowering experiment in rat

The experimental SD rats were acclimatically bred in the laboratory for 1 week (4-week-old SD rats, SPF, male, purchased from Shanghai Silik laboratory animals GmbH. Certification No.: 20170005013017, SCXK 2017-. 7 days before the experiment, the animals are fasted for 4h, then blood is collected in the orbit, and the blood is centrifuged at 3500RPM for 15min to obtain serum. Serum lipid concentrations (triglycerides) were determined using the ADVIA 2400 chemical system (Siemens). Rats were divided into 4 groups (hIgG negative control group, Evinacumab3.5mpk group, P3G3.5mpk group, P3G7mpk group) by triglyceride level, 6 rats in each group were administered once by subcutaneous injection, and on the 1 st, 5 th, 9 th and 13 th days after injection, each group of rats was fasted for 4 hours, and serum was isolated and serum lipid concentration (triglyceride) was measured using the ADVIA 2400 chemical system (Siemens). The experimental data are expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism5 software, statistical analysis was performed using TTEST.

The experimental results are shown in FIG. 2, compared with the hIgG negative control group, triglyceride reduction of each group is reduced after 1 day of administration, and triglyceride reduction of the P3G3.5mpk group and the P3G7mpk group is greater than that of the Evinacumab3.5mpk group; at day 9 post-dose, serum lipid concentrations remained lower for the p3g3.5mpk group and the P3G7mpk group than for the positive control.

Test example 6 determination of the in vivo Effect of anti-ANGPTL 3 antibodies on plasma lipid concentrations in high-fat high-cholesterol fed c57bl/6 mice

The c57bl/6 mice are bred in 5 mice per cage (4-week-old c57bl/6 mice, SPF, about 16-18g, male, purchased from Shanghai Kavens laboratory animal Limited liability company, qualification No. 201913812, SCXK (Su) 2016-. After 5 days of pre-blood collection, the mice were fed with high-fat and high-cholesterol diet (D12079B, available from cooperative medical bioengineering, Inc. of Jiangsu province) instead of normal diet until the end of the experiment. Animals were fasted 8 days before the start of the experiment, orbital bleeding after 4 hours of fasting, centrifuged at 8000RPM for 2min, and plasma was collected. Plasma lipid concentrations were determined using the ADVIA 2400 chemical system (Siemens). Mice were divided into 4 groups (hIgG negative control group, Evinacumab25mpk group, P8BG5mpk group) by triglyceride level, and 11 mice were subcutaneously administered once a week for 8 times. At

weeks

2, 3, 5, 7, 9, 10 and 11 after the start of the experiment, each group of mice was fasted for 4 hours before orbital bleeding and plasma was collected. Plasma lipid concentrations (triglycerides (TG), Total Cholesterol (TC), and LDL cholesterol (LDL-C)) were determined using the ADVIA 2400 chemical system (Siemens). The experimental data are expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism5 software, statistical analysis was performed using TTEST.

The results are shown in tables 8-10 and FIGS. 3-5, and TG, TC and LDL-C were significantly reduced in the P8BG25mpk group after

week

2, 3, 5 and 7 of the experiment compared to the hIgG negative control group; the Evinacumab25mpk and P8BG5mpk groups showed a decrease in TG, TC and LDL-C after 3, 5 and 7 weeks of the experiment. After 9 weeks of the experiment, only the triglycerides of the two groups, P8BG25mpk and P8BG5mpk, were significantly reduced compared to the hIgG negative control group. Triglyceride and total cholesterol were significantly reduced in the P8BG25mpk group at week 2, week 3, week 9 of the experiment compared to the Evinacumab25mpk group; LDL-C was significantly reduced in the P8BG25mpk group at

weeks

2 and 9 of the experiment compared to the Evinacumab25mpk group. In the whole experimental period from the first to 11 weeks, the Evinacumab25mpk group TG had decreased to 40.5 + -2.3, which was 32.60% lower than the starting TG; and the TG of the P8BG25mpk group is reduced to 21.1 +/-1.3, which is reduced by 64.90 percent compared with the starting TG. This indicates that the P8BG antibody is more effective and longer lasting in reducing blood lipids than Evinacumab.

TABLE 8 Effect of anti-ANGPTL 3 antibodies on plasma TG in c57bl/6 mice

Remarking: max Inh% represents the maximum percent lipid lowering relative to the starting TG

TABLE 9 Effect of anti-ANGPTL 3 antibodies on plasma LDL in c57bl/6 mice

Remarking: max Inh% indicates the maximum percentage of lipid lowering relative to LDL in the negative control group

TABLE 10 Effect of anti-ANGPTL 3 antibodies on plasma TC in c57bl/6 mice

Remarking: max Inh% represents the maximum percentage of lipid lowering relative to negative control TC

Test example 7 determination of the in vivo Effect of anti-ANGPTL 3 antibodies on plasma lipid concentrations in APOE mice

APOE mice are bred in 2 cages (APOE mice of 8 weeks old, SPF, about 22g, male, purchased from Shanghai Kavens laboratory animal GmbH, qualification No.: 201917260, SCXK (threo) 2016-. Animals were fasted 7 days before the start of the experiment, orbital bleeding after 4 hours (h) of fasting, centrifuged at 8000RPM for 2 minutes (min), and plasma was collected. Plasma lipid concentrations were determined using the ADVIA 2400 chemical system (Siemens). Mice were divided into 5 groups (hIgG negative control group, Evinacumab10mpk group, Evinacumab5mpk group, P8BG10mpk group, and P8BG5mpk group) according to triglyceride level, 8 mice were each administered once by subcutaneous injection (the administration dose of each group is shown in Table 11), and mice were subjected to orbital bleeding after fasting for 4 hours on day 1 (d), day 7, and day 11 after administration, and plasma lipid concentrations (triglyceride, HDL cholesterol) were measured using ADVIA 2400 chemical system (Siemens). The experimental data are expressed as Mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism5 software, statistical analysis was performed using TTEST.

The results of this experiment are shown in FIGS. 6, 7 and Table 11, and compared with the hIgG negative control group, the triglycerides of the treatment groups were decreased after 1d administration, the triglycerides of the Evincumab (10mpk) group were decreased to 65.6 + -5.4 mg/dL, 46.2% compared with the starting TG value, the triglycerides of the Evincumab (5mpk) group were decreased to 71.8 + -3.3 mg/dL, 42.1% compared with the starting TG value, the triglycerides of the P8BG (10mpk) group were decreased to 33.3 + -2.2 mg/dL, 72.9% compared with the starting TG value, 37.0 + -1.7 mg/dL compared with the P8BG (5mpk) group, and 70.2% compared with the starting TG value. In addition, the experimental results show that the plasma HDL-C of P8BG10mpk group is increased and the plasma HDL-C of Evinacumab10mpk group is reduced after 1 day of administration, while in the lipid-lowering experiment, the reduction of HDL-C is generally not desired and caused at the same time.

TABLE 11 Effect of anti-ANGPTL 3 antibodies on APOE mouse plasma triglycerides

Test example 8 evaluation of pharmacokinetics of anti-ANGPTL 3 antibody

In vivo pharmacokinetic experiment of anti-ANGPTL 3 antibody mice

C57BL/6 mice weighing 18-24g (purchased from Wentonlifwa laboratory animals technologies, Inc., Zhejiang). The laboratory environment adaptive feeding is not less than 3 days, the light/dark period is regulated for 12/12 hours, the temperature is 16-26 ℃, the relative humidity is 40-70%, and the feed and water are freely taken in the feeding period. The day before the start of the experiment, C57BL/6 mice were numbered and randomly grouped into 3 mice each. On the experimental day, mice in each group are respectively injected with test drugs P8BG and Evinacumab intravenously, and the administration dose is 10 mg/kg; the injection volume was 5 ml/kg.

The administration group collected 0.1ml of whole blood before and 5min,8h,1d,2d,4d,7d,10d,14d,21d and 28d after administration, without anticoagulation, placed at 4 ℃ for 30min after blood collection, centrifuged at 1000g for 15min, taken supernatant (serum) in an EP tube, and stored at-80 ℃.

The antibody concentration in the serum was measured by ELISA and the pharmacokinetic parameters of the drug tested were calculated using Winnolin software. The primary pharmacokinetic results obtained are shown in table 12 and figure 8. The experimental results show that the P8BG antibody of the disclosure has a half-life in mice that is higher than that of the positive control, Evinacumab.

TABLE 12 pharmacokinetics of anti-ANGPTL 3 antibodies in mice

Antibodies	P8BG	Evinacumab
Mode of administration	i.v.	i.v.
Administration dose (mg/kg)	10	10
t1/2h	131.4±18.8	71.1±6.1

t1/2d	5.47±0.78	2.96±0.25
AUC0-∞(h*ug/ml)	23272±2887	16904±4481

Remarking: AUC0- ∞: area under the curve when taking medicine; t1/2 (h): half-life (in hours); t1/2 (d): half-life (in days); i.v. intravenous injection.

Second, pharmacokinetic evaluation of anti-ANGPTL 3 antibody in cynomolgus monkey

Cynomolgus monkeys weighing 3-4kg (purchased from Guangdong frontier Biotech Co., Ltd.) were used. The animals are carried out quarantine and domesticated for at least 14 days before the experiment. Animals were housed in stainless steel cages, with no more than 2 animals per cage per group. The room temperature of the animal room is controlled at 18-26 ℃, the relative humidity is 40-70%, and the light and the shade alternate in 12 hours of illumination. Animals were free to ingest water during the experiment. The 6 cynomolgus monkeys were randomly divided into 2 groups of 3 animals each according to biochemical criteria. The test drugs P8BG and Evinacumab are respectively injected into each group subcutaneously, and the administration dose is 10 mg/kg; the injection volume was 2 ml/kg.

After grouping, animals are fasted overnight, and about 2mL of blood is taken from lower limb veins before administration and 1 hour (h), 2h, 4h, 8h, 12h, 1 day (d), 3d, 5d, 7d,10d,14d,21d,28d, 35d, 42d, 49d and 56d after administration starts, the blood is placed in a blood collection tube containing separation gel, the blood is placed at room temperature for 30 minutes (min), 1000g of the blood is centrifuged for 15min, and the supernatant is taken and divided into 2 clean EP tubes to be temporarily stored at the temperature below-70 ℃ (the temporary storage is completed within 2h after blood collection). The antibody concentration in the serum was measured by ELISA and the pharmacokinetic parameters of the drug tested were calculated using Winnolin software. The primary pharmacokinetic results obtained are shown in table 14 and fig. 9. The experimental results show that the P8BG antibodies of the disclosure have a higher half-life in cynomolgus monkeys than the positive control, Evinacumab.

TABLE 13 anti-ANGPTL 3 antibodies in cynomolgus monkey pharmacokinetics

	P8BG	Evinacumab
Administration dose (mg/kg)	10	10
Mode of administration	sc.	sc.
t1/2h	324.7±115.3	157.7±28.6
t1/2d	13.5±4.8	6.6±1.2
AUC 0-∞(ug/ml*h)	44243±7404	38027±252

Remarking: AUC0- ∞: area under the curve when taking medicine; t1/2 (h): half-life (in hours); t1/2 (d): half-life (in days); sc. subcutaneous injection.

Claims

An anti-ANGPTL 3 antibody or antigen-binding fragment thereof, comprising an antibody heavy chain variable region and a light chain variable region, wherein:

i) the heavy chain variable region is similar to the sequence shown in SEQ ID NO:13 having the same sequences as HCDR1, HCDR2 and HCDR3, and a light chain variable region which is substantially identical to the heavy chain variable region as set forth in SEQ ID NO: the light chain variable region shown in the 14 sequence has LCDR1, LCDR2 and LCDR3 with the same sequence;

ii) the heavy chain variable region is substantially identical to the sequence set forth in SEQ ID NO:11 having the same sequences as HCDR1, HCDR2 and HCDR3, and a light chain variable region which is substantially identical to the heavy chain variable region as set forth in SEQ ID NO: 12, the light chain variable region shown in the sequence has LCDR1, LCDR2 and LCDR3 with the same sequence; or

iii) the heavy chain variable region is substantially identical to the sequence set forth in SEQ ID NO:9 having the same sequences as HCDR1, HCDR2 and HCDR3, and a light chain variable region which is substantially identical to the heavy chain variable region as set forth in SEQ ID NO: the light chain variable region shown in the 10 sequence has LCDR1, LCDR2 and LCDR3 with the same sequence.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein:

iv) the heavy chain variable region comprises the sequences shown in SEQ ID NO: 24. SEQ ID NO: 28 and SEQ ID NO: 26, and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 27. SEQ ID NO: 22 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 23;

v) the heavy chain variable region comprises the sequences shown as SEQ ID NO: 24. SEQ ID NO: 25 and SEQ ID NO: 26, and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 21. SEQ ID NO: 22 and SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown at 23; or

vi) the heavy chain variable region comprises the sequences shown in SEQ ID NOs: 18. SEQ ID NO: 19 and SEQ ID NO: 20 and HCDR1, HCDR2 and HCDR3, wherein the light chain variable region comprises the amino acid sequences set forth in SEQ ID NOs: 15. SEQ ID NO: 16 and SEQ ID NO: 17 LCDR1, LCDR2 and LCDR 3.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein:

(A) the heavy chain variable region is identical to SEQ ID NO:13 and/or the light chain variable region has at least 90% sequence identity to SEQ ID NO: 14 have at least 90% sequence identity;

(B) the heavy chain variable region is identical to SEQ ID NO:11 and/or the light chain variable region has at least 90% sequence identity to SEQ ID NO: 12 have at least 90% sequence identity; or

(C) The heavy chain variable region is identical to SEQ ID NO:9 and/or the light chain variable region has at least 90% sequence identity to SEQ ID NO: 10 have at least 90% sequence identity.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to claim 3, comprising a heavy chain variable region and a light chain variable region as shown below:

(D) a heavy chain variable region having a sequence shown in SEQ ID NO 13, and a light chain variable region having a sequence shown in SEQ ID NO: 14, a light chain variable region;

(E) a heavy chain variable region having a sequence shown in SEQ ID NO:11, and a light chain variable region having a sequence shown in SEQ ID NO: 12; or

(F) A heavy chain variable region having a sequence shown in SEQ ID NO 9, and a light chain variable region having a sequence shown in SEQ ID NO: 10, or a light chain variable region.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the anti-ANGPTL 3 antibody further comprises a heavy chain constant region and a light chain constant region;

preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4 constant regions, and the light chain constant region is selected from the group consisting of human kappa and lambda chain constant regions;

more preferably, the heavy chain constant region has the sequence shown as SEQ ID NO. 29 or has at least 85% sequence identity with SEQ ID NO. 29 and/or the light chain constant region has the sequence shown as SEQ ID NO. 30 or has at least 85% sequence identity with SEQ ID NO. 30.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the anti-ANGPTL 3 antibody comprises a heavy chain and a light chain, wherein,

(G) the heavy chain has the same structure as SEQ ID NO: 35 and/or the light chain has at least 85% sequence identity to SEQ ID NO: 36 have at least 85% sequence identity;

(H) the heavy chain has the same structure as SEQ ID NO: 33, and/or the light chain has at least 85% sequence identity to SEQ ID NO: 34 have at least 85% sequence identity; or

(I) The heavy chain has the same structure as SEQ ID NO: 31 and/or the light chain has at least 85% sequence identity to SEQ ID NO: 32 have at least 85% sequence identity.
The anti-ANGPTL 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the anti-ANGPTL 3 antibody comprises a heavy chain and a light chain, wherein,

(J) the heavy chain is shown as SEQ ID NO: 35, and the light chain is as set forth in SEQ ID NO: 36 is shown;

(K) the heavy chain is shown as SEQ ID NO: 33, and the light chain is as set forth in SEQ ID NO: 34; or

(L) the heavy chain is as shown in SEQ ID NO: 31, and the light chain is as set forth in SEQ ID NO: shown at 32.
An isolated anti-ANGPTL 3 antibody or antigen-binding fragment thereof that competes for binding to an ANGPTL3 antigen with the anti-ANGPTL 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 7.
A nucleic acid molecule encoding the anti-ANGPTL 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 8.
A host cell comprising the nucleic acid molecule of claim 9.
A pharmaceutical composition comprising:

(A) a therapeutically effective amount of an anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, and

(B) one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
A method for immunodetection or determination of ANGPTL3, the method comprising the step of using the anti-ANGPTL 3 antibody or antigen binding fragment thereof of any one of claims 1 to 8.
A kit comprising the anti-ANGPTL 3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.
A method of treating a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of the anti-ANGPTL 3 antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the nucleic acid molecule of claim 9, or the pharmaceutical composition of claim 11; preferably, the disease or disorder is an ANGPTL 3-associated disease or disorder; more preferably, the disease or condition is hypercholesterolemia, hyperlipidemia, or atherosclerotic disease.